CN105441411A - Endo cellulase Cel5A mutant with improved performances - Google Patents

Abstract

The present invention relates to mutation of one or more amino acid sites of trichoderma reesei endonuclease Cel5A. A stable trichoderma reesei endonuclease Cel5A thermally stable mutant is designed by rational design and experimental verification method, and the stable mutant can be used in cellulose hydrolysis under high temperature conditions.

Description

A kind of Mierocrystalline cellulose restriction endonuclease Cel5A mutant of enhanced property

Technical field

What the present invention relates to is the 5 family's glycoside hydrolases modified, and is specifically a kind of Mierocrystalline cellulose restriction endonuclease Cel5A mutant at high temperature having enhanced property.

Background technology

Filamentous fungus T. reesei can secrete a large amount of cellulases (>50g/L), is successfully applied to suitability for industrialized production.The cellulase of its secretion mainly comprises: excision enzyme Cel7A, Cel6A; Restriction endonuclease Cel7B, Cel5A, Cel12A, and Cel45A; Polyglucosidase, dextran mono-oxygenase Cel61A(<1%) etc.Except the potential application in bioenergy conversion process, cellulase plays a significant role in animal-feed processing, textile washing, paper pulp processing etc. etc.

Cellulosic high temperature enzymolysis has a lot of advantages, and such as, under high temperature, the reduction of system viscosity allows more charging capacity, and high temperature reduces the risk of microbial contamination, more coordinates with High Temperature Pre process, and hydrolysis rate faster.But the cellulase deriving from Trichodermareesei activity at high temperature and stability all bad (Takashima, Iikuraetal.1998, Lantz, Goedegebuuretal.2010).The expression amount deriving from the high temperature fiber element enzyme of the bacterium usually then unusual end, economy is poor.So the stability improving trichoderma reesei cellulase is necessary.

5 family's glycoside hydrolases are main components of Trichodermareesei Mierocrystalline cellulose restriction endonuclease, knock out this expression of enzymes gene and cause endonuclease activity loss to reach 55% (Suominen, Mantylaetal.1993).Qin etc. find the amino acid mutants N251R(SEQIDNO:1 of Trichodermareesei Cel5A by saturation mutation) optimal pH of enzyme can be become 6.2 from 4.8, and improve its activity (Qin at a high ph further by orthogenesis, Weietal.2008, Qin, Weietal.2008).US Patent No. 2013/0095554A1 finds that mutant G293A/S can improve the activity 1.2 times of this enzyme.

The raising of protein stability can have been come by orthogenesis or design and rational.The method that design and rational adopts usually comprises: the core improving protein piles up (DahiyatandMayo1997), remove the polar group (Blaber buried, Lindstrometal.1993, Hendsch, Jonssonetal.1996), change surface charging group distribution (Makhatadze, Loladzeetal.2003, Gribenko, Pateletal.2009, SchweikerandMakhatadze2009), disulfide linkage (RobinsonandSauer2000) etc. is introduced.

The thermostability of protein is very important for it in the application of biological technical field.Such as in industrial enzyme, good thermostability means survival time more of a specified duration and the temperature of reaction of Geng Gao.And in pharmaceutical grade protein, good thermostability then ensure that more lasting survival time and drug effect.

Summary of the invention

The object of the invention is to provide a kind of Mierocrystalline cellulose restriction endonuclease Cel5A mutant at high temperature having enhanced property.

For achieving the above object, the technical solution used in the present invention is:

1. a Mierocrystalline cellulose restriction endonuclease Cel5A mutant for enhanced property, it is characterized in that: the amino acid alignment of described mutant and SEQIDNO:1, its mutational site comprises N38D, V102I, D217A, D271L, and one or more in D271M.

2. an application for the Mierocrystalline cellulose restriction endonuclease Cel5A mutant of enhanced property according to claim 1, is characterized in that: described mutant can be used as catalyzer for catalyzed reaction.

The advantage that the present invention has:

1. the thermostability of the present invention by using design and rational to improve Cel5A, stable mutant can be used to do high-temperature catalytic.

Accompanying drawing explanation

The Cel5A that Fig. 1 provides for the embodiment of the present invention and mutant thereof are to the graph of a relation between CMC residual activity and holding temperature.With sigmoid function, nonlinear fitting is carried out to it, and calculate temperature corresponding when residual activity is 50%, namely t ₅₀.

The building process of the recombinant plasmid pET-22b-cel5A-CD that Fig. 2 provides for the embodiment of the present invention; Wherein 1. extract the total serum IgE of Trichodermareesei and utilize RT-PCR to obtain the cDNA library of this mould; with this cDNA library for masterplate and known Cel5A gene order design primer; obtained the gene order of Cel5A by Standard PCR, and be cloned into plasmid pUC19 formation pUC19-cel5A; 2. be masterplate with pUC19-cel5A, cel5A-CD gene fragment is obtained by Standard PCR, then cut with NdeI and XhoI enzyme with pET-22b simultaneously, rubber tapping is reclaimed rear alcohol settling product and is adopted T4 ligase enzyme to connect, transform and be coated with LB flat board, choose transformant to cultivate, upgrading grain sequence verification, and preserve corresponding recombinant plasmid.

Embodiment

The present invention is by from Trichodermareesei restriction endonuclease Cel5A crystalline structure, (the pdb:3QR3), the unstable site potential to crystalline structure is selected, and introduces the interaction under mutant increase folded state subsequently.The mutant of demonstration validation thermostability raising by experiment, and the activity of mutant is characterized.

Below in conjunction with embodiment, method of the present invention is described further, wherein the test method of unreceipted actual conditions, usually can conditional operation routinely.

Embodiment 1

The selection of design and rational mutant:

The present invention is by from restriction endonuclease Cel5A crystalline structure, (the pdb:3QR3), adopts structure analysis software DiscoveryStudio to analyze structure, finds that D271 is the polare Aminosaeren of part embedding, be unfavorable for protein stabilization, and being mutated into A, L, M can eliminate pendant polar; V102 is embedding hydrophobic amino acid, and there is hole its side chain region, increases side-chain bulk and should contribute to improving stability; N38 side chain and K32 form hydrogen bond, N38 is mutated into aspartic acid and then this effect may be become salt bridge, be conducive to stablizing.Molecular biology method is adopted to carry out Experimental Characterization to these mutant subsequently.

Embodiment 2

1. the generation of expression vector and protein:

1) first to Trichodermareesei ( trichodermareeseiqM9414) carry out enlarged culturing and collect thalline, then its total serum IgE is extracted, RT-PCR method is utilized to build the cDNA library of Trichodermareesei, secondly with this cDNA for the method that masterplate utilizes T to clone transfers Mierocrystalline cellulose restriction endonuclease Cel5A gene, and be cloned in plasmid pUC19, after sequence verification, by correct Cel5A catalytic domain (catalyticdomain ,cel5A -cD) gene is through pcr amplification, then cuts with NdeI and XhoI enzyme with pET-22b simultaneously, and rubber tapping is reclaimed rear alcohol settling product and adopted T4 ligase enzyme to connect, and transforms and is coated with LB flat board, upgrading grain sequence verification, and preserve recombinant plasmid pET-22b- cel5A-cD (see Fig. 2).

2) according to calculating the Cel5A-CD mutational site and password sublist design mutant primer determined, and with pET-22b- cel5A-cD is the method recombinant C el5A-CD mutant plasmid that template utilizes inverse PCR, and wherein amplification condition is: (1.95 DEG C, 5min; 94 DEG C, 30s, 60 DEG C, 30s, 72 DEG C, 6min30s, 30 circulations; 72 DEG C, 10min; 4 DEG C of insulations).

3) above-mentioned each amplification PCR primer reclaimed by fragment rubber tapping, phosphorylation, phosphorylation after product adopts the connection of T4 ligase enzyme to spend the night, after transforming, be coated with LB flat board, chooses transformant and cultivates, and then gets the order-checking of bacterium liquid.

The above-mentioned Cel5A-CD mutational site design primer determined is shown in (table 1):

Table 1.Cel5A mutant primer sequence table

2. Activity determination:

The 280nm place optical extinction coefficient of Cel5A-CD is that 67880(obtains (http://web.expasy.org/protparam/) according to Cel5A-CD amino acid composition by online tool); By the absorption value Quantitative Western concentration of each mutant protein solution 280nm of Cel5A-CD and above-mentioned acquisition, each mutant does three parallel measurements, record 280nm absorbance value.Then formulae discovery is passed through: protein concentration=A ₂₈₀/ optical extinction coefficient, the mean value of three panel datas is the protein concentration of Cel5A-CD and Cel5A-CD mutant.Equivalent protein concentration is used to detect to compare Cel5A-CD and its mutant for the activity of substrate carboxymethyl sodium cellulosate (CMC) and phosphoric acid swollen cellulose (PASC), the active method of PAHBAH that adopts detects (Lever1972), be specially the solution preparing concentration from low to high with glucose, then add isopyknic different glucose and PAHBAH working fluid reacts, measure A with ultraviolet spectrophotometer ₄₁₀light absorption value and do typical curve.It is 0.05 μm of ol/L that enzymic catalytic reaction system comprises enzyme concn, the 50mM Acetic acid-sodium acetate reaction solution of the CMC of 0.1%, pH5.0, and 50 DEG C of reaction 5min, detect by PAHBAH method and calculate the amount of the reducing sugar that reaction produces according to typical curve.Be 200 μ L to the detection catalyst system of PASC activity, enzyme concn is 1 μm of ol/L, concentration of substrate is 5mg/ml, pH5.050mM Acetic acid-sodium acetate damping fluid, 50 DEG C of reaction 10min, reaction solution is through 0.45 μm of membrane filtration, extract reaction solution and detect (Lever1972) by PAHBAH method, and calculating enzymic activity according to typical curve, enzyme work is defined as the catalysis 1 micromole substrate enzyme amount be converted into needed for product in per minute and is defined as enzyme activity unit, i.e. a 1U.The results are shown in Table 2.

The expression activitiy of table 2.Cel5A-CD wild-type and mutant and different substrate reactions

3.Tm matching

After making Cel5A-CD and Cel5A-CD mutant be incubated 5min from low temperature to high temperature under (63 DEG C-81 DEG C) condition, detect each enzyme and substrate CMC is reacted to the residual activity retained under 50 DEG C of conditions, getting Cel5A-CD and its mutant the activity of 50 DEG C is 100%, and the residual activity under each temperature condition is done sigmoid matching (WuandArnold2013), temperature when calculated activity is 50%, namely t ₅₀(Fig. 1), concrete outcome is in table 3.Wherein in the mutant of experimental verification, D271L, D271M, D271A, V102I, N38D mutant demonstrates higher t ₅₀numerical value (table 3).

The Tm value of table 3.Cel5A-CD wild-type and single disulfide linkage mutant.

Cel5A-CD	T 50（℃）	Improve temperature (DEG C)
			WT	72.2
D271L	75.0	2.8
			D271M	74.6	2.4
D271A	73.2	1.0
			N38D	72.3	0.1
V102I	72.5	0.3

Reference:

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Dahiyat,B.I.andS.L.Mayo(1997)."Probingtheroleofpackingspecificityinproteindesign." ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica 94( 19):10172-10177.

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SEQIDNO:1(WT)

1MGVRFAGVNIAGFDFGCTTD

21GTCVTSKVYPPLKNFTGSNN

41YPDGIGQMQHFVNDDGMTIF

61RLPVGWQYLVNNNLGGNLDS

81TSISKYDQLVQGCLSLGAYC

101IVDIHNYARWNGGIIGQGGP

121TNAQFTSLWSQLASKYASQS

141RVWFGIMNEPHDVNINTWAA

161TVQEVVTAIRNAGATSQFIS

181LPGNDWQSAGAFISDGSAAA

201LSQVTNPDGSTTNLIFDVHK

221YLDSDNSGTHAECTTNNIDG

241AFSPLATWLRQNNRQAILTE

261TGGGNVQSCIQDMCQQIQYL

281NQNSDVYLGYVGWGAGSFDS

301TYVLTETPTGSGNSWTDTSL

321VSSCLARKGGSGSGHHHHHH

341HHH*

SEQIDNO:2(N38D)

1MGVRFAGVNIAGFDFGCTTD

21GTCVTSKVYPPLKNFTGSNN

41YPDGIGQMQHFVNDDGMTIF

61RLPVGWQYLVNNNLGGNLDS

81TSISKYDQLVQGCLSLGAYC

101IVDIHNYARWNGGIIGQGGP

121TNAQFTSLWSQLASKYASQS

141RVWFGIMNEPHDVNINTWAA

161TVQEVVTAIRNAGATSQFIS

181LPGNDWQSAGAFISDGSAAA

201LSQVTNPDGSTTNLIFDVHK

221YLDSDNSGTHAECTTNNIDG

241AFSPLATWLRQNNRQAILTE

261TGGGNVQSCIQDMCQQIQYL

281NQNSDVYLGYVGWGAGSFDS

301TYVLTETPTGSGNSWTDTSL

321VSSCLARKGGSGSGHHHHHH

341HHH*

SEQIDNO:3(V102I)

1MGVRFAGVNIAGFDFGCTTD

21GTCVTSKVYPPLKNFTGSNN

41YPDGIGQMQHFVNDDGMTIF

61RLPVGWQYLVNNNLGGNLDS

81TSISKYDQLVQGCLSLGAYC

101IVDIHNYARWNGGIIGQGGP

121TNAQFTSLWSQLASKYASQS

141RVWFGIMNEPHDVNINTWAA

161TVQEVVTAIRNAGATSQFIS

181LPGNDWQSAGAFISDGSAAA

201LSQVTNPDGSTTNLIFDVHK

221YLDSDNSGTHAECTTNNIDG

241AFSPLATWLRQNNRQAILTE

261TGGGNVQSCIQDMCQQIQYL

281NQNSDVYLGYVGWGAGSFDS

301TYVLTETPTGSGNSWTDTSL

321VSSCLARKGGSGSGHHHHHH

341HHH*

SEQIDNO:4(D271L)

1MGVRFAGVNIAGFDFGCTTD

21GTCVTSKVYPPLKNFTGSNN

41YPDGIGQMQHFVNDDGMTIF

61RLPVGWQYLVNNNLGGNLDS

81TSISKYDQLVQGCLSLGAYC

101IVDIHNYARWNGGIIGQGGP

121TNAQFTSLWSQLASKYASQS

141RVWFGIMNEPHDVNINTWAA

161TVQEVVTAIRNAGATSQFIS

181LPGNDWQSAGAFISDGSAAA

201LSQVTNPDGSTTNLIFDVHK

221YLDSDNSGTHAECTTNNIDG

241AFSPLATWLRQNNRQAILTE

261TGGGNVQSCIQLMCQQIQYL

281NQNSDVYLGYVGWGAGSFDS

301TYVLTETPTGSGNSWTDTSL

321VSSCLARKGGSGSGHHHHHH

341HHH*

SEQIDNO:5(D271M)

1MGVRFAGVNIAGFDFGCTTD

21GTCVTSKVYPPLKNFTGSNN

41YPDGIGQMQHFVNDDGMTIF

61RLPVGWQYLVNNNLGGNLDS

81TSISKYDQLVQGCLSLGAYC

101IVDIHNYARWNGGIIGQGGP

121TNAQFTSLWSQLASKYASQS

141RVWFGIMNEPHDVNINTWAA

161TVQEVVTAIRNAGATSQFIS

181LPGNDWQSAGAFISDGSAAA

201LSQVTNPDGSTTNLIFDVHK

221YLDSDNSGTHAECTTNNIDG

241AFSPLATWLRQNNRQAILTE

261TGGGNVQSCIQMMCQQIQYL

281NQNSDVYLGYVGWGAGSFDS

301TYVLTETPTGSGNSWTDTSL

321VSSCLARKGGSGSGHHHHHH

341HHH*

SEQIDNO:6(D271A)

1MGVRFAGVNIAGFDFGCTTD

21GTCVTSKVYPPLKNFTGSNN

41YPDGIGQMQHFVNDDGMTIF

61RLPVGWQYLVNNNLGGNLDS

81TSISKYDQLVQGCLSLGAYC

101IVDIHNYARWNGGIIGQGGP

121TNAQFTSLWSQLASKYASQS

141RVWFGIMNEPHDVNINTWAA

161TVQEVVTAIRNAGATSQFIS

181LPGNDWQSAGAFISDGSAAA

201LSQVTNPDGSTTNLIFDVHK

221YLDSDNSGTHAECTTNNIDG

241AFSPLATWLRQNNRQAILTE

261TGGGNVQSCIQAMCQQIQYL

281NQNSDVYLGYVGWGAGSFDS

301TYVLTETPTGSGNSWTDTSL

321VSSCLARKGGSGSGHHHHHH

341HHH*

Claims (2)

1. a Mierocrystalline cellulose restriction endonuclease Cel5A mutant for enhanced property, it is characterized in that: the amino acid alignment of described mutant and SEQIDNO:1, its mutational site comprises N38D, V102I, D217A, D271L, and one or more in D271M.

2. an application for the Mierocrystalline cellulose restriction endonuclease Cel5A mutant of enhanced property according to claim 1, is characterized in that: described mutant can be used as catalyzer for catalyzed reaction.